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The heterogeneity in venom composition and potency in disparate Eastern Russell's viper (Daboia siamensis) populations has repercussions for the efficacy of antivenoms. This is particularly pronounced in geographical areas in which the venom of the local species has not been well studied and locally produced antivenoms are unavailable. In such cases, alternative therapies following envenoming, which are not limited by species specificity, may be employed to complement antivenoms. We studied the neuromuscular activity of D. siamensis venom from Thailand and Java (Indonesia) and the ability of Thai antivenoms and/or Varespladib to prevent or reverse these effects. Both Thai and Javanese D. siamensis venoms displayed potent pre-synaptic neurotoxicity but weak myotoxicity in the chick biventer cervicis nerve–muscle preparation. Whilst the neurotoxicity induced by both venoms was abolished by the prior administration of Thai D. siamensis monovalent antivenom or pre-incubation with Varespladib, Thai neuro-polyvalent antivenom only produced partial protection when added prior to venom. Pre-synaptic neurotoxicity was not reversed by the post-venom addition of either antivenom 30 or 60 min after either venom. Varespladib, when added 60 min after venom, prevented further inhibition of indirect twitches. However, the subsequent addition of additional concentrations of Varespladib did not result in further recovery from neurotoxicity. The combination of Thai monovalent antivenom and Varespladib, added 60 min after venom, resulted in additional recovery of twitches caused by either Thai or Javanese venoms compared with antivenom alone. In conclusion, we have shown that Varespladib can prevent and partially reverse the pre-synaptic neurotoxicity induced by either Thai or Javanese D. siamensis venoms. The efficacy of Thai D. siamensis monovalent antivenom in reversing pre-synaptic neurotoxicity was significantly enhanced by its co-administration with Varespladib. Further work is required to establish the efficacy of Varespladib as a primary or adjunct therapy in human envenoming. [ABSTRACT FROM AUTHOR]

The synaptic protein–DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA–protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific–specific (synaptic), non-specific–non-specific (non-specific), and specific–non-specific (pre-synaptic) SfiI–DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein–DNA complexes discovered in the time-lapse AFM experiments. [ABSTRACT FROM AUTHOR]

Spontaneous subthreshold activity in the central nervous system is fundamental to information processing and transmission, as it amplifies and optimizes sub-threshold signals, thereby improving action potential initiation and maintaining reliable firing. This form of spontaneous activity, which is frequently considered noise, is particularly important at auditory synapses where acoustic information is encoded by rapid and temporally precise firing rates. In contrast, when present in excess, this form of noise becomes detrimental to acoustic information as it contributes to the generation and maintenance of auditory disorders such as tinnitus. The most prominent contribution to subthreshold noise is spontaneous synaptic transmission (synaptic noise). Although numerous studies have examined the role of synaptic noise on single cell excitability, little is known about its pre-synaptic modulation owing in part to the difficulties of combining noise modulation with monitoring synaptic release. Here we study synaptic noise in the auditory brainstem dorsal cochlear nucleus (DCN) of mice and show that pharmacological potentiation of Kv3 K+ currents reduces the level of synaptic bombardment onto DCN principal fusiform cells. Using a transgenic mouse line (SyG37) expressing SyGCaMP2-mCherry, a calcium sensor that targets pre-synaptic terminals, we show that positive Kv3 K+ current modulation decreases calcium influx in a fifth of pre-synaptic boutons. Furthermore, while maintaining rapid and precise spike timing, positive Kv3 K+ current modulation increases the synchronization of local circuit neurons by reducing spontaneous activity. In conclusion, our study identifies a unique pre-synaptic mechanism which reduces synaptic noise at auditory synapses and contributes to the coherent activation of neurons in a local auditory brainstem circuit. This form of modulation highlights a new therapeutic target, namely the pre-synaptic bouton, for ameliorating the effects of hearing disorders which are dependent on aberrant spontaneous activity within the central auditory system.

Spontaneous subthreshold activity in the central nervous system is fundamental to information processing and transmission, as it amplifies and optimizes sub-threshold signals, thereby improving action potential initiation and maintaining reliable firing. This form of spontaneous activity, which is frequently considered noise, is particularly important at auditory synapses where acoustic information is encoded by rapid and temporally precise firing rates. In contrast, when present in excess, this form of noise becomes detrimental to acoustic information as it contributes to the generation and maintenance of auditory disorders such as tinnitus. The most prominent contribution to subthreshold noise is spontaneous synaptic transmission (synaptic noise). Although numerous studies have examined the role of synaptic noise on single cell excitability, little is known about its pre-synaptic modulation owing in part to the difficulties of combining noise modulation with monitoring synaptic release. Here we study synaptic noise in the auditory brainstem dorsal cochlear nucleus (DCN) of mice and show that pharmacological potentiation of Kv3 K+ currents reduces the level of synaptic bombardment onto DCN principal fusiform cells. Using a transgenic mouse line (SyG37) expressing SyGCaMP2-mCherry, a calcium sensor that targets pre-synaptic terminals, we show that positive Kv3 K+ current modulation decreases calcium influx in a fifth of pre-synaptic boutons. Furthermore, while maintaining rapid and precise spike timing, positive Kv3 K+ current modulation increases the synchronization of local circuit neurons by reducing spontaneous activity. In conclusion, our study identifies a unique pre-synaptic mechanism which reduces synaptic noise at auditory synapses and contributes to the coherent activation of neurons in a local auditory brainstem circuit. This form of modulation highlights a new therapeutic target, namely the pre-synaptic bouton, for ameliorating the effects of hearing disorders which are dependent on aberrant spontaneous activity within the central auditory system. [ABSTRACT FROM AUTHOR]

Les Neuropathies Auditives (NA) représentent environ 10 % des surdités de perception de l'enfant. Elles correspondent à un défaut de traitement du signal acoustique par le nerf cochléaire. La surdité en cas de NA peut être congénitale ou d'apparition secondaire, stable ou évolutive, isolée ou syndromique. La surdité est de degré variable, l'intelligibilité de la parole est dégradée, plus qu'attendue. Ces manifestations sont majorées en condition d'écoute bruyante, avec d'éventuelles fluctuations. Il s'agit d'un groupe hétérogène dont le diagnostic repose sur un faisceau d'arguments cliniques et électrophysiologiques ainsi que sur la discordance de certains éléments : persistance de l'activité des cellules ciliées externes et tracés des potentiels évoqués auditifs du tronc cérébral altérés ; discordance entre les seuils de l'audiométrie tonale et le degré d'intelligibilité de la parole. Il existe deux principaux modes de présentation des NA chez l'enfant : (1) La période néonatale : surdité congénitale d'origine génétique ; surdité acquise (ex : ictère néonatal, très grande souffrance néonatale...) ; (2) La pré-adolescence : pathologie évolutive principalement d'origine génétique avec dégradation progressive de l'intelligibilité et possible poly-neuropathies associées. Le bilan audiométrique doit comporter une audiométrie tonale et vocale ainsi qu'un enregistrement de l'activité des cellules ciliées externes et des potentiels évoqués du tronc cérébral. Il est important de préciser au mieux le site lésionnel grâce au bilan génétique et à l'imagerie, compte tenu des implications thérapeutiques et pronostiques. Auditory Neuropathy Spectrum Disorder (ANSD) accounts for around 10% of pediatric endocochlear deafness. ANSD results from a failure in the processing of acoustic stimuli by the cochlear nerve. Deafness in the event of ANSD can be congenital or secondary, stable or progressive, isolated or syndromic. Deafness is of varying degrees, and intelligibility is degraded, more than expected. These symptoms are increased in noisy conditions, with possible fluctuations. Individuals with ANSD form a heterogeneous group. A diagnosis is established based on results from a battery of clinical and electrophysiological tests, as well as on discrepancies in certain elements: persistence of outer hair cell (OHC) activity and impairment of auditory brainstem response (ABR), differences between audiometric threshold and speech recognition. There are two main presentations of ANSD in children: (1) The neonatal period: congenital deafness of genetic origin ; acquired deafness (eg neonatal jaundice, severe neonatal injury, etc...); (2) Pre-adolescence: progressive pathology with deterioration of intelligibility, possibly associated with poly-neuropathies, mainly of genetic origin. The audiometric assessment must include tonal and speech audiometry as well as a recording of both the activity of the outer hair cell and the auditory brainstem response. Because of the therapeutic and prognostic implications, it is important to specify the site of the lesion as much as possible through genetic evaluation and imaging. [ABSTRACT FROM AUTHOR]

Purpose: We examined the effect of ischemic preconditioning (IPC) on changes in muscle force, activation, and the spinal reflex pathway during and after repeated sprint cycling.Methods: Eight recreationally active men (high-intensity cardiorespiratory training > 3 times per week, > 6 months) completed two exercise sessions (5 sets of 5 cycling sprints, 150% max W), preceded by either IPC (3 × 5 min leg occlusions at 220 mmHg) or SHAM (3 × 5 min at 20 mmHg). Knee extensor maximal force and rate of force were measured before (PRE), immediately post (POST), 1H, and 24H after cycling. Twitch interpolation and resting potentiated twitches were applied to estimate voluntary activation and muscle contractility, respectively. Quadriceps H-reflex recruitment curves were collected at all time-points using 10 Hz doublet stimulation to allow estimation of H-reflex post-activation depression. Surface electromyograms and tissue oxygenation (via near-infrared spectroscopy) were continuously recorded during cycling.Results: IPC did not affect any measure of neuromuscular function or performance during cycling. Maximal force and muscle contractility were significantly lower at POST and 1H compared to PRE and 24H by up to 50% (p < 0.01). Maximal force was lower than PRE at 24H by 8.7% (p = 0.028). Voluntary activation and rate of force were unchanged. A rightwards shift was observed for the H-reflex recruitment curve POST, and post-activation depression was higher than all other time-points at 24H (p < 0.05). Muscle activation and oxygenation decreased during cycling.Conclusions: IPC has a nominal effect on mechanisms associated with neuromuscular function during and after exercise in healthy populations. [ABSTRACT FROM AUTHOR]

Behavioral genetic studies of humans have associated variation in the DTNBP1 gene with schizophrenia and its cognitive deficit phenotypes. The protein coded for by DTNBP1, dysbindin, is expressed within forebrain glutamatergic neurons, in which it interacts with proteins involved in vesicular trafficking and exocytosis. In order to further delineate the cellular, physiological, and behavioral phenotypes associated with reduced dysbindin expression, we conducted studies in mice carrying a null mutation within the dtnbp1 gene. Dysbindin mutants showed impairments of spatial working memory compared with wild-type controls; heterozygous mice showed intermediate levels of cognitive dysfunction. Deep-layer pyramidal neurons recorded in the prefrontal cortex of mutant mice showed reductions in paired-pulse facilitation, and evoked and miniature excitatory post-synaptic currents, indicating a difference in the function of pre-synaptic glutamatergic terminals as well as elevated spike thresholds. Taken together, these data indicate that dysbindin potently regulates excitatory transmission in the prefrontal cortex, potentially through a pre-synaptic mechanism, and consequently modulates cognitive functions depending on this brain region, providing new insights into the molecular mechanisms underlying cortical dysfunction in schizophrenia.

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

The prevalence and importance of hearing damage caused by noise levels not previously thought to cause permanent hearing impairment has become apparent in recent years. The damage to, and loss of, afferent terminals of auditory nerve fibres at the cochlear inner hair cell has been well established, but the effects of noise exposure and terminal loss on the inner hair cell are less known. Using three-dimensional structural studies in mice we have examined the consequences of afferent terminal damage on inner hair cell morphology and intracellular structure. We identified a structural phenotype in the pre-synaptic regions of these damaged hair cells that persists for four weeks after noise exposure, and demonstrates a specific dysregulation of the synaptic vesicle recycling pathway. We show evidence of a failure in regeneration of vesicles from small membrane cisterns in damaged terminals, resulting from a failure of separation of small vesicle buds from the larger cisternal membranes.

Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity. [ABSTRACT FROM AUTHOR]

The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins. [ABSTRACT FROM AUTHOR]

Russell's vipers are snakes of major medical importance in Asia. Russell's viper ( Daboia russelii) envenoming in Sri Lanka and South India leads to a unique, mild neuromuscular paralysis, not seen in other parts of the world where the snake is found. This study aimed to identify and pharmacologically characterise the major neurotoxic components of Sri Lankan Russell's viper venom. Venom was fractionated using size exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). In vitro neurotoxicities of the venoms, fractions and isolated toxins were measured using chick biventer and rat hemidiaphragm preparations. A phospholipase A (PLA) toxin, U1-viperitoxin-Dr1a (13.6 kDa), which constitutes 19.2 % of the crude venom, was isolated and purified using HPLC. U1-viperitoxin-Dr1a produced concentration-dependent in vitro neurotoxicity abolishing indirect twitches in the chick biventer nerve-muscle preparation, with a t of 55 ± 7 min only at 1 μM. The toxin did not abolish responses to acetylcholine and carbachol indicating pre-synaptic neurotoxicity. Venom, in the absence of U1-viperitoxin-Dr1a, did not induce in vitro neurotoxicity. Indian polyvalent antivenom, at the recommended concentration, only partially prevented the neurotoxic effects of U1-viperitoxin-Dr1a. Liquid chromatography mass spectrometry analysis confirmed that U1-viperitoxin-Dr1a was the basic S-type PLA toxin previously identified from this venom (NCBI-GI: 298351762; SwissProt: P86368). The present study demonstrates that neurotoxicity following Sri Lankan Russell's viper envenoming is primarily due to the pre-synaptic neurotoxin U1-viperitoxin-Dr1a. Mild neurotoxicity observed in severely envenomed Sri Lankan Russell's viper bites is most likely due to the low potency of U1-viperitoxin-Dr1a, despite its high relative abundance in the venom. [ABSTRACT FROM AUTHOR]

Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. [ABSTRACT FROM AUTHOR]

Munc13-1 is a pre-synaptic active-zone protein essential for neurotransmitter release and involved in pre-synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13-1 with EC50s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13-1 C1 exclusively at Glu-582, which was identified by mass spectrometry. Mutation of Glu-582 to alanine, leucine, and histidine reduced the alcohol binding two- to five-fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild-type Munc13-1 compared with the mutants. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Dunc-13 in Drosophila melanogaster. The Dunc-13 P84200/+ heterozygotes have 50% wild-type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. The present studies indicate that Munc13-1 C1 has binding site(s) for alcohols and Munc13-1 activity is sufficient to restore normal self-administration to Drosophila mutants deficient in Dunc-13 activity. [ABSTRACT FROM AUTHOR]

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor ( KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca2+ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca2+ entry through Ca2+-permeable KARs and liberation of intracellular Ca2+ stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca2+] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca2+-calmodulin dependent activation of an adenylyl cyclase/ cAMP/protein kinase A signalling cascade, independent of G-protein involvement. [ABSTRACT FROM AUTHOR]

Apolipoprotein E ( APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock-in mice, we have previously shown that APOE-ε4 Targeted Replacement ( TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post-synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate-glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE-ε3 TR mice, APOE-ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE-ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre-synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4-5 months and 1 year. Using high-frequency 13C/1H nuclear magnetic resonance spectroscopy, we found that APOE-ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE-ε4, and also act as surrogate markers for Alzheimer's disease risk. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 113, 1611–1620. We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Nav) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Nav coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [3H]glutamate and [14C]GABA release to the specific Nav blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Nav subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Nav subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Nav and Nav1.2. Nerve terminal-specific differences in the expression of specific Nav subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release. [ABSTRACT FROM AUTHOR]

Biochemical and physiological evidence suggest that pre-synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that ‘Rab3a Interacting Molecule’ (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co-immunoprecipitation (co-IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti-RIM antibody, pRIM1,2: while staining for the two proteins co-localized and co-varied at the transmitter release face, consistent with an association, they failed to co-IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel-tethering variant. We find that co-immunostaining with mRIM and anti-CaV2.2 antibody neither co-localized nor co-varied at the transmitter release face and the two proteins did not co-IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre-fusion secretory vesicle to the calcium channel protein complex. [ABSTRACT FROM AUTHOR]

Abstract: The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory and gustatory information. In the present study, patch-clamp electrophysiological experiments were conducted using rat horizontal brainstem sections. Pre-synaptic and somatic/dendritic nicotinic acetylcholine receptors (nAChRs) expressed in neurons of the caudal NTS (cNTS) were found to be randomly distributed between pre-synaptic and somatic/dendritic sites (χ2 =0.72, df=3, p >0.87, n =200). Pre-synaptic nAChRs were detected by their facilitating effects on glutamatergic neurotransmission of a sub-population of cNTS neurons (categorized as “effect-positive”) upon brief picospritzer applications of 0.1–0.5mM nicotine. These effects were resistant to inhibition by 20nM methyllycaconitine (MLA) and 4μM dihydro-β-erythroidine (DHβE), and were replicated by brief picospritzer applications of 0.2–1mM cytisine. Picospritzer applications of 0.2mM RJR-2403, a potent agonist of α4β2 nAChRs, did not facilitate synaptic release of glutamate in effect-positive cNTS neurons. The population of somatic/dendritic nAChRs has been found to be heterogeneous and included nAChRs that were activated by RJR-2403 and/or cytisine, or insensitive to cytisine, or inhibited by MLA. The presented results are consistent with the expression of β4-containing (i.e., β4*) nAChRs, likely α3β4*, in pre-synaptic terminals of effect-positive cNTS neurons. Somatic/dendritic nAChRs appear to involve both α7 and non-α7 subunits. Heterogeneity in the subunit composition of pre-synaptic and somatic/dendritic nAChRs may underlie diverse roles that these receptors play in regulation of behavioral and visceral reflexes, and may reflect specific targeting by endogenous nicotinic agents and nicotine. [Copyright &y& Elsevier]

Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre- and post-synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post-synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre- and post-synaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the post-synaptic membrane. We have also demonstrated that neurexin Iβ is largely (96%) pre-synaptic. Thus, neurexin Iβ and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals. [ABSTRACT FROM AUTHOR]

In the brain, the polyamines spermidine (Spd) and spermine (Spm) serve highly specific functions by interacting with various ion channel receptors intimately involved with synaptic signaling. Both, glial cells and neurons contain Spd/Spm, but release and uptake mechanisms could re-distribute polyamines between cell types. The cellular and subcellular localization of polyamine biosynthetic enzymes may therefore offer a more appropriate tool to identify local sources of enhanced Spd/Spm synthesis, which may be related with specific roles in neuronal circuits and synaptic function. A recently characterized antibody against Spd synthase was therefore used to screen the rat brain for compartment-specific peaks in enzyme expression. The resulting labeling pattern indicated a clearly heterogeneous expression predominantly localized to neurons and neuropil. The highest levels of Spd synthase expression were detected in the accumbens nucleus, taenia tecta, cerebellar cortex, cerebral cortical layer I, hippocampus, hypothalamus, mesencephalic raphe nuclei, central and lateral amygdala, and the circumventricular organs. Besides a diffuse labeling of the neuropil in several brain areas, the distinct labeling of mossy fiber terminals in the cerebellar cortex directly indicated a synaptic role for Spd synthesis. Electron microscopy revealed a preferential distribution of the immunosignal in synaptic vesicle containing areas. A pre-synaptic localization was also observed in parallel and climbing fiber terminals. Electrophysiological recordings in acute cerebellar slices revealed a Spd-induced block of evoked extracellular field potentials resulting from mossy fiber stimulation in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation. [ABSTRACT FROM AUTHOR]

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons. [Copyright &y& Elsevier]

The effects of three protein kinase C (PKC) agonists (phorbol ester, ingenol and indolactam-V) and two PKC antagonists (d-erythro-sphingosine and chelerythrine) on input–output (I–O) relations in the Schaffer collateral pathway to CA1 (SC-CA1) and mossy fiber pathway to CA3 (MF-CA3) were determined in rat hippocampus brain slices. In the SC-CA1 pathway, phorbol esters and indolactam-V had only small effects on field excitatory post-synaptic potentials (fEPSP) in slices from 60-day animals, although ingenol, an activator of novel PKC isozymes, caused a significant decrease of the field excitatory post-synaptic potentials amplitude in 60-day animals, but not in 30-day animals. In contrast, in the MF-CA3 pathway, PKC agonists induced a significant increase in the field excitatory post-synaptic potentials. PKC antagonists depressed the field excitatory post-synaptic potentials in the SC-CA1 pathway, but had no significant effect in the MF-CA3 pathway. In the MF-CA3 pathway, paired-pulse facilitation was abolished by PKC agonists and unaffected by antagonists. In SC-CA1, it was depressed by agonists to levels below control, whereas it was significantly increased by chelerythine. We conclude that PKC plays important but different roles in both regions. In the SC-CA1 pathway, PKC is almost maximally active under control circumstances, and PKC antagonists significantly reduce synaptic responses. In contrast, in the MF-CA3 pathway, there is no apparent activation under resting circumstances, but significant potentiation of synaptic transmission is induced when PKC is activated. There are developmental changes in the pattern of PKC isozymes, and both pre- and post-synaptic actions are important. [Copyright &y& Elsevier]

Abstract In this study we have tested the effects of a wide range of metabotropic glutamate receptor ligands on (i) depolarisation-evoked efflux of pre-accumulated d-[[sup 3] H]aspartic acid (d-[[sup 3] H]asp) from rapidly superfused rat cerebrocortical minislices, and (ii) Na[sup +] -dependent uptake of d-[[sup 3] H]asp into cerebrocortical tissue. Transient elevations in extracellular K[sup +] produced concentration-dependent increases in d-[[sup 3] H]asp efflux. A submaximally effective concentration (50 mm) was used in all subsequent experiments. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; EC[sub 50] 17.8 μm], the group I mGlu-selective agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG; EC[sub 50] 0.5 μm] and the mGlu5 receptor subtype-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine [(RS)-CHPG; EC[sub 50] 7.3 μm] all concentration-dependently potentiated high K[sup +] -evoked d-[[sup 3] H]asp efflux in the absence of effects on basal outflow of radiolabel. At concentrations selective for mGlu1 receptors, the antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid [(RS)-AIDA; 10–300 μm]; (+)-2-methyl-4-carboxyphenylglycine [LY367385; 1–100 μm] and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylate ethyl ester [CPCCOEt, 1–30 μm] all failed to inhibit responses to (S)-3,5-DHPG. However, the broad-spectrum mGlu receptor antagonist (S)-α-methyl-4-carboxyphenylglycine [(S)-MCPG; IC[sub 50] 88.5 μm] together with the recently described mGlu5-selective antagonists, 2-methyl-6-(phenylethynyl)-pyridine (MPEP; IC[sub 50] 0.6 μm), 6-methyl-2-(phenyl-azo)-3-pyridinol (SIB-1757; IC[sub 50] 4.4 μm) and (E )-2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893; IC[sub 50] 3.1 μm), at mGlu5-selective concentrations, all powerfully and concentration-dependently inhibited (S)-3,5-DHPG-evoked responses. Two selective excitatory amino acid (EAA) uptake inhibitors, l-trans -2,4-pyrrolidine dicarboxylate (l-trans -2,4-PDC; IC[sub 50] 229 μm) and dl-threo -β-benzyloxyaspartate (dl-TBOA; IC[sub 50] 665 μm) both inhibited the Na[sup +] -dependent uptake of d-[[sup 3] H]asp into cerebrocortical minislices. Importantly, none of the mGlu ligands utilized in the present study significantly inhibited d-[[sup 3] H]asp uptake at concentrations shown to potentiate K[sup +] -evoked efflux. These data demonstrate for the first time that mGlu5 ligands modulate extracellular EAA concentrations by a direct effect on mGlu5-type autoreceptors on EAA nerve terminals as they evoke clear changes in EAA release in the absence of any effects on EAA uptake. Selective mGlu5 receptor antagonists that show high potency and good central bioavailability may provide novel classes of neuroprotective agents for the treatment of brain disorders associated with abnormal EAAergic neurotransmission. [ABSTRACT FROM AUTHOR]

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [[sup 35]S]methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture. [ABSTRACT FROM AUTHOR]

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals. [ABSTRACT FROM AUTHOR]

Background: This study used novel human neurophysiologic models to investigate whether the mechanism of rate-sensitive H-reflex depression lies in the pre-synaptic or post-synaptic locus in humans. We hypothesized that pre-synaptic inhibition would suppress Ia afferents and H-reflexes without suppressing alpha motor neurons or motor evoked potentials (MEPs). In contrast, post-synaptic inhibition would suppress alpha motor neurons, thereby reducing H-reflexes and MEPs., Methods: We recruited 23 healthy adults with typical rate-sensitive H-reflex depression, 2 participants with acute sensory-impaired spinal cord injury (SCI) (to rule out influence of sensory stimulation on supra-spinal excitability), and an atypical cohort of 5 healthy adults without rate-sensitive depression. After a single electrical stimulation to the tibial nerve, we administered either a testing H-reflex or a testing MEP at 50-5000 ms intervals., Results: Testing MEPs were not diminished in healthy subjects with or without typical rate-sensitive H-reflex depression, or in subjects with sensory-impaired SCI. MEP responses were similar in healthy subjects with versus without rate-sensitive H-reflex depression., Conclusions: Results from these novel in vivo human models support a pre-synaptic locus of rate-sensitive H-reflex depression for the first time in humans. Spinal reflex excitability can be modulated separately from descending corticospinal influence. Each represents a potential target for neuromodulatory intervention., (Copyright © 2019 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Muscles innervated by the facial nerve show differential sensitivities to muscle relaxants than muscles innervated by somatic nerves. The evoked electromyography (EEMG) response is also proportionally reduced after facial nerve injury. This forms the theoretical basis for proper utilization of muscle relaxants to balance EEMG monitoring and immobility under general anesthesia. (1) To observe the relationships between the level and mode of acetylcholine (ACh) release and the duration of facial nerve injury, and the influence of rocuronium in an in vitro rabbit model. (2) To explore the pre-synaptic mechanisms of discrepant responses to a muscle relaxant. Quantal and non-quantal ACh release were measured by using intracellular microelectrode recording in the orbicularis oris 1 to 42 days after graded facial nerve injury and in the gastrocnemius with/without rocuronium. Quantal ACh release was significantly decreased by rocuronium in the orbicularis oris and gastrocnemius, but significantly more so in gastrocnemius. Quantal release was reduced after facial nerve injury, which was significantly correlated with the severity of nerve injury in the absence but not in the presence of rocuronium. Non-quantal ACh release was reduced after facial nerve injury, with many relationships observed depending on the extent of the injury. The extent of inhibition of non-quantal release by rocuronium correlated with the grade of facial nerve injury. These findings may explain why EEMG amplitude might be diminished after acute facial nerve injury but relatively preserved after chronic injury and differential responses in sensitivity to rocuronium.